The present invention relates to treatment and prevention of hyperproliferative conditions in humans and more particularly to antisense oligonucleotides complementary to human replication-initiation genes which modulate DNA replication and cell proliferation. This invention also relates to methods of using such oligonucleotides to inhibit the growth of tumor cells in mammals.
Cancer presents one of the most serious threats to human health and life. There are approximately ten million new cancer cases and seven million cancer-related deaths every year in the world. In fact, approximately one in every four people has the probability of developing cancer during their lifetime.
Some examples of anticancer drugs currently available are cytotoxins, DNA damaging agents, and inhibitors of oncogenic proteins involved in signal transduction pathways for cell proliferation. However, few of the current anticancer drugs are effective or without side effects. Many of these drugs are not highly selective towards cancer cells and these drugs also damage normal cells or inhibit the metabolism and cellular functions of normal cells. Moreover, most oncogenic signal transduction pathways are redundant in the cells, therefore treatments that block individual pathways can be bypassed by cancer cells and inhibition of one or some of these pathways may not stop cancer growth.
Antisense oligonucleotides are known to be able to inhibit gene expression, and have been used in combination with chemotherapeutic agents to develop anticancer strategies in mouse xenographs (Geiger, T. et al., Anti-Cancer Drug Design, 13, 35-45 (1998), Del Bufalo, D. et al, British J. of Cancer, 74, 387-393 (1996)). Current theories suggest that the activity of antisense oligonucleotides depends on the binding of the oligonucleotides to the target nucleic acid (e.g. to at least a portion of a genomic region, gene or mRNA transcript thereof), thus disrupting the function of the target, either by hybridization arrest or by destruction of target RNA by RNase H (activated by nucleotide hybridization to RNA). For example, antisense oligonucleotides bind to the complementary sequence on a target mRNA nucleic acid sequence, thus activating endogenous RNase H to cleave mRNA. Binding of antisense oligonucleotides to mRNA may also interfere with translation thereby reducing or eliminating production of a gene even if the mRNA is not degraded (Milligan, J. F., et al, J. Med. Chem., 36, 1923-1927 (1993)). However, known antisense oligonucleotides have not targeted human replication-initiation genes, nor demonstrated any efficacy in inhibiting expression of replication-initiation proteins in human cells.
Proteins involved in the initiation of DNA replication (i.e., genome duplication) present excellent targets for cancer therapy. Initiation of DNA replication is controlled by the cis-acting DNA elements called replicators and the trans-acting initiation proteins that interact with the replicators. To date, several groups of initiation proteins required for eukaryotic DNA replication have been identified. These include ORC (origin recognition complex), Cdc6 (cell division cycle), MCM (minichromosome maintenance), Cdc45 and Cdt1 proteins (Takisawa, H., et al., Curr. Opin. Cell. Biol. 12, 690-696 (2000)). ORC binds chromatin throughout the cell cycle, whereas the chromatin association of other groups is cell cycle-regulated (Leatherwood, J., Curr. Opin. Cell. Biol., 10, 742-748 (1998)). Some of the initiation proteins in humans, such as Cdc6 and MCM proteins, are expressed in cancerous, but not in normal, non-dividing cells (Williams, G. H. et al., Proc. Natl. Acad. Sci. USA, 95, 14932-14937 (1998)). Gene sequences encoding some of these proteins have been isolated (Williams, U.S. Pat. No. 5,851,821; Saha, P. et al., J. Biol. Chem., 50, 6075-6086 (1990); Todorov, L T., et al., J. Cell Sci., 107, 253-265 (1994)).